Recombinant antigen aiming at Zika / dengue virus as well as vaccine composition and application thereof

Abstract

The invention relates to a recombinant antigen aiming at Zika / dengue virus as well as a vaccine composition and application thereof. The recombinant antigen is obtained by introducing specific mutation into an FL fusion loop region and optionally a non-FL fusion loop region of an E protein of the wild type Zika / dengue virus; by introducing specific mutation, the FL epitope of the recombinant antigen is destroyed, and the generation of an induced FL epitope antibody can be avoided, so that the aim of reducing or eliminating an ADE effect is fulfilled; moreover, the recombinant antigen has correct E protein conformation, has protein expression quantity similar to or higher than that of a wild type, and can induce relatively high specific binding antibody titer and neutralizing antibody titer in animal bodies, so that effective immune protection can be provided, and the recombinant antigen has relatively good clinical application value and industrialization prospect.

Description

[0001] Cross-reference

[0002] This application claims the priority of a Chinese patent application with the application number 202410024322.3, titled "Recombinant Antigens Against Zika / Dengue Viruses, Their Vaccine Compositions and Applications", filed on January 5, 2024, the entire content of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and specifically to recombinant antigens against Zika / Dengue viruses, their vaccine compositions and applications. Background Art

[0004] Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus of the family Flaviviridae. It was first discovered in the jungles of Uganda, Africa. However, there are still no vaccines or drugs available so far. Although the global incidence of ZIKV infection has decreased, ZIKV still poses a threat to people living in endemic areas. Therefore, the development of a ZIKV vaccine is urgent.

[0005] Dengue virus (DV) is the pathogen of dengue fever (DF), which is mainly transmitted by vector insects such as Aedes aegypti and Aedes albopictus and is widely distributed in tropical and subtropical regions. The structures of ZIKV and DV viruses are relatively similar. Both are spherical, have an envelope, and the envelope surface contains envelope (E) protein. The internal viral genome is single-stranded positive-sense RNA, about 11 kb in length, and contains only one open reading frame. The polyprotein translated therefrom can be cleaved into 3 structural proteins (C, prM, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). Among them, the C protein constitutes the nucleocapsid; when the virus matures, the PrM protein is enzymatically cleaved to form the membrane protein M and then fixed to the inner layer of the virus envelope. It is an important co-component for the virus to induce protective immunity and also helps the correct folding of the E protein and the stability of its structure; the E protein is the main glycoprotein of the virus envelope and is related to the cell tropism of the virus, hemagglutination, and the induction of the production of hemagglutination-inhibiting antibodies and neutralizing antibodies. It is the main protective antigen of Zika or Dengue virus.

[0006] The E protein exists in the form of a dimer. Each monomer has three domains, namely DI, DII, and DIII. Among them, the head of DII (amino acids 98 - 109) contains a highly conserved fusion loop region (FL). The FL region sequences of ZIKV and DV viruses are exactly the same, both being D98 - R99 - G100 - W101 - G102 - N103 - G104 - C105 - G106 - L107 - F108 - G109. The FL region plays a key role in the membrane fusion process during virus invasion. During the virus infection process, immune cells will produce a large number of antibodies against FL.

[0007] DV belongs to the genus Flavivirus in the family Flaviviridae and is divided into four serotypes, 1, 2, 3, and 4, according to different antigenicity. DV infection has a typical antibody - dependent infection enhancement (ADE) phenomenon. When a virus of one serotype, such as DV - 1, infects, the body's immune cells will produce corresponding anti - DV - 1 antibodies. However, if infected again with another type of dengue virus, such as DV - 2, the previously produced anti - DV - 1 antibodies not only cannot neutralize the DV - 2 virus but also exacerbate the infection.

[0008] Currently, there are Dengvaxia from Sanofi Pasteur and DVax from Takeda Pharmaceutical Company of Japan, which was just approved this year. The development ideas of the two are similar, both being live attenuated vaccines. The difference is that Dengvaxia uses the yellow fever virus attenuated strain 17D as the vaccine virus backbone, while DVax uses the DV - 2 attenuated strain PDK - 53 as the vaccine virus backbone. DVax can enhance the immune response and protection against DV - 2, but the protection against DV - 3 and 4 is weak. The vaccine Dengvaxia has shown significant ADE safety problems in people without a history of dengue fever infection, so the use of this vaccine is strictly restricted to people with a history of dengue fever infection. In the newly announced 5 - year follow - up data after vaccination this year, the DVax vaccine has also shown potential ADE safety risks in people without a history of dengue fever infection.

[0009] Numerous studies have shown that the hydrophobic fusion loop (FL) of the E protein is the main epitope inducing the ADE effect and also a major dominant epitope on the E protein. Among the antibodies screened from dengue-infected individuals, 41% of the antibodies (46 / 112) target FL (Dejnirattisai W. et al. Nature Immunology, (2016)). To completely eliminate the ADE effect of the vaccine, the usual approach is to mutate the FL epitope so that it cannot induce FL antibodies. At the same time, the mutation should not destroy the remaining epitopes of the E protein or cause a significant decrease in protein expression to ensure that the vaccine can induce sufficient neutralizing antibodies and generate immune protection. However, the FL epitope plays an important role in inducing the fusion of the viral envelope and the host cell membrane when flaviviruses invade host cells. Its amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109 is highly conserved throughout the flavivirus genus. In the previous studies of the inventors, when W101 was mutated to any of the other 25 amino acids, the E protein was hardly expressed; there are only a very few variant sequences in the FL of insect-specific flaviviruses that are distantly related to dengue / zika viruses throughout the flavivirus genus. However, when these variant sequences are applied to dengue vaccines, the mutation results in a significant decrease in E protein expression, and the vaccine cannot provide effective immune protection.

[0010] In view of the above problems existing in the prior art, there is an urgent need to develop a Zika / dengue vaccine that can avoid the generation of the ADE effect and at the same time has high immunogenicity. Summary of the Invention

[0011] Object of the Invention

[0012] Aiming at the problems or needs existing in the prior art, the purpose of the present invention is to provide a Zika / dengue virus recombinant antigen that can avoid the ADE effect, can be efficiently expressed at the same time and has high immunogenicity, a vaccine composition based on the recombinant antigen, and its application.

[0013] Solution

[0014] To achieve the above object, in the process of culturing Zika virus, the present invention adds FL epitope antibody to directionally induce mutations in the FL region of the virus; since the amino acid sequence of the FL epitope of Zika virus is exactly the same as that of the FL epitope of dengue virus, the inventor then introduced the point mutations that occurred in the FL epitope and other positions of Zika virus into the dengue vaccine, and obtained a recombinant E protein antigen against Zika virus / dengue virus. The obtained recombinant E protein antigen against Zika virus / dengue virus can be highly expressed, has high immunogenicity against Zika virus / dengue virus, and can avoid the induction of the production of FL epitope antibody, thereby achieving the effect of reducing or eliminating the ADE effect.

[0015] Specifically, the present invention provides the following technical solutions:

[0016] In the first aspect, the present invention provides a recombinant antigen, the recombinant antigen has the full-length sequence of the E protein of Zika virus or dengue virus, and the E protein has site mutations selected from the following:

[0017] Mutation at position G106 in the FL fusion loop region;

[0018] Double-site mutations at positions W101 and G106 in the FL fusion loop region;

[0019] Double-site mutations at positions W101 and G106 in the FL fusion loop region and mutation of the 125th amino acid in the non-FL fusion loop region;

[0020] Double-site mutations at positions G102 and G106 in the FL fusion loop region;

[0021] Double-site mutations at positions N103 and G106 in the FL fusion loop region;

[0022] Triple-site mutations at positions W101, N103 and G106 in the FL fusion loop region.

[0023] In a preferred embodiment, the mutation at position G106 in the FL fusion loop region is: G106V mutation;

[0024] In a preferred embodiment, the double-site mutations at positions W101 and G106 in the FL fusion loop region are selected from: (1) W101R and G106V, (2) W101G and G106V, and (3) W101L and G106V;

[0025] In a preferred embodiment, the double-site mutations at positions W101 and G106 in the FL fusion loop region and the mutation of the 125th amino acid in the non-FL fusion loop region are: double-site mutations at positions W101R and G106V in the FL fusion loop region and mutation of the 125th amino acid in the non-fusion loop region to valine;

[0026] In a preferred embodiment, the double-site mutation of the FL fusion loop regions G102 and G106 is: the double-site mutation of G102R and G106V;

[0027] In a preferred embodiment, the double-site mutations of the FL fusion loop regions N103 and G106 are selected from: (1) N103T and G106V, (2) N103H and G106V, (3) N103K and G106V, (4) N103P and G106V, and (5) N103Y and G106V;

[0028] In a preferred embodiment, the triple-site mutations of the FL fusion loop regions W101, N103 and G106 are selected from: (1) W101G, N103T and G106V, and (2) W101G, N103K and G106V.

[0029] In some preferred specific embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 1, and the E protein has site mutations selected from the following:

[0030] G106V mutation;

[0031] W101R and G106V double-site mutation;

[0032] W101G and G106V double-site mutation;

[0033] W101L and G106V double-site mutation;

[0034] W101R, G106V and L125V triple-site mutation;

[0035] G102R and G106V double-site mutation;

[0036] N103T and G106V double-site mutation;

[0037] N103H and G106V double-site mutation;

[0038] N103K and G106V double-site mutation;

[0039] N103P and G106V double-site mutation;

[0040] N103Y and G106V double-site mutation;

[0041] W101G, N103T and G106V triple-site mutation;

[0042] W101G, N103K and G106V triple-site mutation.

[0043] In some other preferred specific embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 2, and the E protein has site mutations selected from the following:

[0044] G106V mutation;

[0045] W101R and G106V double-site mutations;

[0046] W101G and G106V double-site mutations;

[0047] W101L and G106V double-site mutations;

[0048] W101R, G106V and M125V triple-site mutations;

[0049] G102R and G106V double-site mutations;

[0050] N103T and G106V double-site mutations;

[0051] N103H and G106V double-site mutations;

[0052] N103K and G106V double-site mutations;

[0053] N103P and G106V double-site mutations;

[0054] N103Y and G106V double-site mutations;

[0055] W101G, N103T and G106V triple-site mutations;

[0056] W101G, N103K and G106V triple-site mutations.

[0057] In some other preferred specific embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, and the E protein has site mutations selected from the following:

[0058] G106V mutation;

[0059] W101R and G106V double-site mutations;

[0060] W101G and G106V double-site mutations;

[0061] W101L and G106V double-site mutations;

[0062] W101R, G106V and I125V triple-site mutations;

[0063] G102R and G106V double-site mutations;

[0064] Double-site mutations of N103T and G106V;

[0065] Double-site mutations of N103H and G106V;

[0066] Double-site mutations of N103K and G106V;

[0067] Double-site mutations of N103P and G106V;

[0068] Double-site mutations of N103Y and G106V;

[0069] Triple-site mutations of W101G, N103T and G106V;

[0070] Triple-site mutations of W101G, N103K and G106V.

[0071] In some other preferred specific embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, and the E protein has site mutations selected from the following:

[0072] G106V mutation;

[0073] Double-site mutations of W101R and G106V;

[0074] Double-site mutations of W101G and G106V;

[0075] Double-site mutations of W101L and G106V;

[0076] Triple-site mutations of W101R, G106V and I125V;

[0077] Double-site mutations of G102R and G106V;

[0078] Double-site mutations of N103T and G106V;

[0079] Double-site mutations of N103H and G106V;

[0080] Double-site mutations of N103K and G106V;

[0081] Double-site mutations of N103P and G106V;

[0082] Double-site mutations of N103Y and G106V;

[0083] Triple-site mutations of W101G, N103T and G106V;

[0084] Triple-site mutations of W101G, N103K and G106V.

[0085] Further preferably, when the recombinant antigen has the full-length sequence of the E protein of Zika virus, the recombinant antigen further comprises the full-length or partial prM protein sequence or M protein sequence of Zika virus.

[0086] Further preferably, when the recombinant antigen has the full-length sequence of the E protein of dengue virus, the recombinant antigen further comprises the full-length or partial prM protein sequence or M protein sequence of the corresponding serotype of dengue virus;

[0087] In a further preferred specific embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 1, the recombinant antigen has the amino acid sequence as shown in SEQ ID NO: 2-14;

[0088] In a further preferred specific embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 2, the recombinant antigen has the amino acid sequence as shown in SEQ ID NO: 16-28;

[0089] In a further preferred specific embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, the recombinant antigen has the amino acid sequence as shown in SEQ ID NO: 30-42;

[0090] In a further preferred specific embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, the recombinant antigen has the amino acid sequence as shown in SEQ ID NO: 44-56.

[0091] In a second aspect, the present invention provides a method for preparing the recombinant antigen as described in the first aspect above, the preparation method comprising the following steps:

[0092] Add a Kozak sequence and the coding sequence of a signal peptide to the 5' end of the nucleotide sequence encoding the recombinant antigen as described in any one of claims 1-8, add the coding sequence of a histidine tag and a stop codon to the 3' end, perform cloning and expression, screen for correct recombinants, and then transfect the cells of the expression system for expression, collect the cell culture supernatant, and isolate the recombinant antigen therefrom.

[0093] Preferably, the cells of the expression system are mammalian cells, insect cells, yeast cells or bacterial cells;

[0094] Optionally, the mammalian cells are HEK293T cells, 293F series cells or CHO cells; further optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells;

[0095] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells;

[0096] Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom;

[0097] Optionally, the bacterial cells are Escherichia coli cells.

[0098] In a feasible embodiment, the signal peptide can be any signal peptide commonly used in the art; for example, the signal peptide can be a signal peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 57-61.

[0099] In a third aspect, the present invention provides a polynucleotide encoding the recombinant antigen as described in the first aspect above. The polynucleotide is not limited by the method for producing it. For example, it can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0100] The polynucleotide can be DNA or mRNA, preferably mRNA.

[0101] In a preferred specific embodiment, the polynucleotide is DNA having a sequence as shown in any one of SEQ ID NOs: 62-113, or mRNA corresponding to the DNA.

[0102] Specifically, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 2-14, the polynucleotide encoding the recombinant antigen is DNA having a sequence as shown in SEQ ID NOs: 62-74, or mRNA corresponding to the DNA; when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 16-28, the polynucleotide encoding the recombinant antigen is DNA having a sequence as shown in SEQ ID NOs: 75-87, or mRNA corresponding to the DNA; and / or, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 30-42, the polynucleotide encoding the recombinant antigen is DNA having a sequence as shown in SEQ ID NOs: 88-100, or mRNA corresponding to the DNA; and / or, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 44-56, the polynucleotide encoding the recombinant antigen is DNA having a sequence as shown in SEQ ID NOs: 101-113, or mRNA corresponding to the DNA.

[0103] The above-mentioned "mRNA corresponding to the DNA" refers to an mRNA having such a sequence, that is, an RNA sequence formed by replacing all "T"s in the specified DNA sequence with "U"s.

[0104] Fourth aspect, the present invention provides an expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus comprising the polynucleotide as described in the third aspect above.

[0105] Fifth aspect, the present invention provides a vaccine or immunogenic composition, which comprises the recombinant antigen as described in the first aspect above, or the polynucleotide as described in the third aspect above, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus as described in the fourth aspect above as an active ingredient.

[0106] In a specific embodiment, in addition to the active ingredient, the vaccine or immunogenic composition further comprises a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0107] In some feasible embodiments, the vaccine or immunogenic composition is a recombinant protein vaccine, which comprises the recombinant antigen as described in the first aspect above and an adjuvant;

[0108] Preferably, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, CpG adjuvant, QS-21-containing adjuvant, AS series adjuvants (such as AS01 series adjuvants) and nanoparticle adjuvants.

[0109] In some other feasible embodiments, the vaccine or immunogenic composition is a DNA vaccine, and the DNA vaccine comprises:

[0110] (i) a eukaryotic expression vector; and

[0111] (ii) a DNA sequence encoding the recombinant antigen as described in the first aspect above, which is constructed into the eukaryotic expression vector;

[0112] Preferably, the DNA sequence encoding the recombinant antigen as described in the first aspect above is a DNA sequence shown in any one of SEQ ID NOs: 62-113;

[0113] Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0114] In some other feasible embodiments, the vaccine or immunogenic composition is an mRNA vaccine, and the mRNA vaccine comprises:

[0115] (I) an mRNA sequence encoding the recombinant antigen as described in the first aspect above; and

[0116] (II) a lipid nanoparticle;

[0117] Preferably, the mRNA sequence encoding the recombinant antigen as described in the first aspect above is the mRNA sequence corresponding to the DNA sequence shown in any one of SEQ ID NOs: 62-113.

[0118] In some other feasible embodiments, the vaccine or immunogenic composition is a viral vector vaccine, which includes:

[0119] (1) a viral backbone vector; and

[0120] (2) a DNA sequence encoding the recombinant antigen as described in the first aspect above, which is constructed into the viral backbone vector;

[0121] Preferably, the DNA sequence encoding the recombinant antigen as described in the first aspect above is the DNA sequence shown in any one of SEQ ID NOs: 62-113;

[0122] Preferably, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, lentivirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

[0123] In a feasible implementation manner, the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;

[0124] Preferably, the nasal spray is selected from aerosols, sprays and powder aerosols;

[0125] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated tablets and ointments;

[0126] More preferably, the tablet is a sublingual tablet;

[0127] More preferably, the granule is a fine granule;

[0128] More preferably, the powder is a powder for external use;

[0129] More preferably, the pill is a small pill;

[0130] Preferably, the parenteral preparation is a transdermal agent, ointment, plaster, external liquid preparation, injectable preparation; more preferably, the injectable preparation is a pushable preparation.

[0131] In the sixth aspect, the present invention provides the use of the recombinant antigen as described in the first aspect above, or the polynucleotide as described in the third aspect above, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus as described in the fourth aspect above in the preparation of a drug for detecting, preventing and / or treating Zika virus or dengue virus infection.

[0132] Preferably, the drug is a vaccine.

[0133] In a seventh aspect, the present invention provides a method for preventing and / or treating Zika virus or dengue virus infection, the method comprising: administering to a subject in need a prophylactically and / or therapeutically effective amount of the following substances: the recombinant antigen as described in the first aspect above, the polynucleotide as described in the third aspect above, the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus as described in the fourth aspect above, and / or the vaccine or immunogenic composition as described in the fifth aspect above.

[0134] The "prophylactically and / or therapeutically effective amount" may vary depending on the administration subject, target organ, symptoms, administration method, etc., and can be determined according to the doctor's judgment by considering the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, etc.

[0135] Advantageous Effects

[0136] The recombinant antigen of the present invention against Zika / dengue virus effectively eliminates the FL epitope in the E protein, avoiding the generation of antibodies against the FL epitope that cause ADE, thus significantly reducing or eliminating the ADE effect; at the same time, the recombinant antigen can maintain the correct conformation of the E protein and has a protein expression level similar to or higher than that of the wild type. It has been confirmed by animal immunization experiments that the Zika / dengue vaccine based on this recombinant antigen can induce high specific antibody titers and neutralizing antibody titers in animals, thus providing effective immune protection, and the cross-antibody titers against E proteins of other serotypes induced by it are significantly reduced, so the ADE risk of the vaccine is also greatly reduced.

[0137] In addition, the vaccine obtained based on the recombinant antigen provided by the present invention can include various forms, such as recombinant protein vaccines, nucleic acid vaccines (such as mRNA vaccines), viral vector vaccines (such as adenovirus vector vaccines), virus attenuated or inactivated vaccines based on the antigen sequence, chimeric vaccines of other backbones, etc., and can be used to prepare Zika and / or dengue vaccines that eliminate the ADE effect, having good clinical application value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] One or more embodiments are illustrated by way of example in the accompanying drawings corresponding thereto, and these exemplary illustrations do not constitute a limitation on the embodiments. The special word "exemplary" used here means "serving as an example, embodiment or illustrative". Any embodiment illustrated as "exemplary" here need not be construed as being superior to or better than other embodiments.

[0139] Figure 1 Showing the antigen epitope detection results of the DV1 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0140] Figure 2 Show the antigenic epitope detection results of the DV2 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0141] Figure 3 Show the antigenic epitope detection results of the DV3 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0142] Figure 4 Show the antigenic epitope detection results of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0143] Figure 5 Show the specific antibody titers against the viral E protein induced by each DV mRNA vaccine, as detected by enzyme-linked immunosorbent assay, as detected in Example 6.

[0144] Figure 6 Show the neutralizing antibody titers against the corresponding serotype DV virus induced by each DV mRNA vaccine, as detected by virus micro-neutralization assay, as detected in Example 7.

[0145] Figure 7 Show the cross-antibody titers against the E proteins of DV2 / 3 / 4 and Zika virus induced by the DV1 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0146] Figure 8 Show the cross-antibody titers against the E proteins of DV1 / 3 / 4 and Zika virus induced by the DV2 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0147] Figure 9 Show the cross-antibody titers against the E proteins of DV1 / 2 / 4 and Zika virus induced by the DV3 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0148] Figure 10 Show the cross-antibody titers against the E proteins of DV1 / 2 / 3 and Zika virus induced by the DV4 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0149] Figure 11 Show the neutralizing antibody titers against the corresponding serotype DV virus induced by the DV1-EM3-1 and DV2-EM3 mRNA vaccines, as detected by virus micro-neutralization assay, as detected in Example 9.

[0150] Figure 12Shows the cross - antibody titers against the E proteins of DV2 / 3 / 4 and Zika virus induced by the DV1 - EM3 - 1 mRNA vaccine prepared in Example 1, as detected in Example 9.

[0151] Figure 13 Shows the cross - antibody titers against the E proteins of DV1 / 3 / 4 and Zika virus induced by the DV2 - EM3 mRNA vaccine prepared in Example 1, as detected in Example 9.

[0152] Figure 14 Shows the ADE effect at the cellular level against DV2 / 3 / 4 virus induced by the immune serum of the DV1 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0153] Figure 15 Shows the ADE effect at the cellular level against DV1 / 3 / 4 virus induced by the immune serum of the DV2 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0154] Figure 16 Shows the ADE effect at the cellular level against DV1 / 2 / 4 virus induced by the immune serum of the DV3 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0155] Figure 17 Shows the ADE effect at the cellular level against DV1 / 2 / 3 virus induced by the immune serum of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0156] Figure 18 Shows the in - vivo ADE effect against DV2 virus induced by the immune serum of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 11.

[0157] Figure 19 Shows the antibody level against the FL epitope induced by the tetravalent dengue mRNA vaccine, detected using the mouse serum of the tetravalent dengue mRNA vaccine obtained in Example 12, as detected in Example 14.

[0158] Figure 20 Shows the neutralizing antibody titers against the four serotype DV viruses induced by the tetravalent dengue mRNA vaccine, detected using the mouse serum of the tetravalent dengue mRNA vaccine obtained in Example 12, as detected in Example 15.

[0159] Figure 21 Shows the challenge protection effects against DV1 and DV2 viruses induced by the tetravalent dengue mRNA vaccine described in Example 12, as detected in Example 16.

[0160] Figure 22 Show the antigen epitope and antigen expression detection results of the DV1 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0161] Figure 23 Show the antigen epitope and antigen expression detection results of the DV2 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0162] Figure 24 Show the antigen epitope and antigen expression detection results of the DV3 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0163] Figure 25 Show the antigen epitope and antigen expression detection results of the DV4 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0164] Figure 26 Show the challenge protection effect against DV2 virus induced by the DV2 mRNA vaccine prepared in Example 17, as detected in Example 19.

[0165] Figure 27 Show the SDS-PAGE results of the dengue virus-like particles prepared in Example 20, as detected in Example 20.

[0166] Figure 28 Show the negative staining results of the DV1-EM3-1 virus-like particles prepared in Example 20 by electron microscopy, as detected in Example 21.

[0167] Figure 29 Show the negative staining results of the DV2-EM3-1 virus-like particles prepared in Example 20 by electron microscopy, as detected in Example 21.

[0168] Figure 30 Show the negative staining results of the DV3-EM3 virus-like particles prepared in Example 20 by electron microscopy, as detected in Example 21.

[0169] Figure 31 Show the negative staining results of the DV4-EM3 virus-like particles prepared in Example 20 by electron microscopy, as detected in Example 21.

[0170] Figure 32 Show the neutralizing antibody titers against the corresponding serotype DV virus induced by each DV recombinant protein vaccine, as detected by the virus micro-neutralization assay in Example 22.

[0171] Figure 33Shows the neutralizing antibody titers against four serotypes of DV virus induced by the tetravalent DV recombinant protein vaccine as detected by the virus micro-neutralization assay, as detected in Example 23. Detailed implementation mode

[0172] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0173] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation mode. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0174] Unless otherwise specified, the implementation of the present invention will adopt conventional techniques in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are all within the technical scope of this field.

[0175] In addition, unless otherwise clearly defined in other parts of this article, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the field to which the present invention belongs. To make the present invention easier to understand, some scientific and technical terms are specifically defined as follows.

[0176] The term "comprising" or its variations such as "including" or "including with" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0177] The term "about", when used in conjunction with numerical values, means to cover numerical values within a range having a lower limit 5% smaller and an upper limit 5% larger than the specified numerical value, including but not limited to ±5%, ±2%, ±1% and ±0.1%, because these variations are suitable for carrying out the disclosed methods.

[0178] The term "and / or" should be understood to mean any one of the options or any combination of any two or more of the options.

[0179] As used herein, the term "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted inclusively, that is, including at least one in the list of quantities or elements, but also including more than one, and optionally, additional unlisted items.

[0180] Example 1: Preparation of Dengue Virus mRNA Vaccine

[0181] In this example, a series of mRNA constructs encoding the full-length prME protein of dengue virus (i.e., including prM protein + E protein) were designed, including:

[0182] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein as shown in SEQ ID NO:1;

[0183] DV1-EM3, encoding the prME antigen protein as shown in SEQ ID NO:3;

[0184] DV1-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:4;

[0185] DV2-WT (i.e., DV2 wild type), encoding the prME antigen protein as shown in SEQ ID NO:15;

[0186] DV2-EM3, encoding the prME antigen protein as shown in SEQ ID NO:17;

[0187] DV2-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:18;

[0188] DV3-WT (i.e., DV3 wild type), encoding the prME antigen protein as shown in SEQ ID NO:29;

[0189] DV3-EM3, encoding the prME antigen protein as shown in SEQ ID NO:31;

[0190] DV3-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:32;

[0191] DV4-WT (i.e., DV4 wild type), encoding the prME antigen protein as shown in SEQ ID NO:43;

[0192] DV4-EM3, encoding the prME antigen protein as shown in SEQ ID NO:45;

[0193] DV4-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:46.

[0194] Next, according to the codon preference of mammalian cells, the nucleic acid sequence encoding the above recombinant dengue virus prME antigen protein was optimized to obtain an optimized nucleic acid coding sequence. Among them, the optimized nucleic acid coding sequences of DV1-WT, DV2-WT, DV3-WT, and DV4-WT are shown in SEQ ID NO: 114-117 respectively, and the optimized nucleic acid coding sequences of DV1-EM3, DV1-EM3-1, DV2-EM3, DV2-EM3-1, DV3-EM3, DV3-EM3-1, DV4-EM3, and DV4-EM3-1 are shown in SEQ ID NO: 63, 64, 76, 77, 89, 90, 102, and 103 respectively; then, the Kozak sequence and the nucleic acid coding sequence of the signal peptide (such as SEQ ID NO: 118, encoding the signal peptide with the amino acid sequence shown in SEQ ID NO: 58) were added to the 5' end of each nucleic acid coding sequence, and a stop codon was added to its 3' end. Then, GenScript Biotech Corporation was commissioned for gene synthesis, and the synthesized gene fragment was recombined onto the pHRNT vector (patent for invention ZL202110224383.0) stored in the laboratory to obtain the template plasmid for preparing the mRNA vaccine.

[0195] The specific preparation process of the mRNA vaccine refers to the method disclosed in the patent for invention ZL202110224383.0. Briefly, the above template plasmid was linearized by enzymatic digestion, and then T7 transcriptase was used for in vitro transcription of mRNA. During the transcription process, uridine was replaced with pseudouridine or N1-methylpseudouridine, etc., to obtain modified mRNA to improve the protein expression level of mRNA in vivo; subsequently, the mRNA was packaged into nanoparticles by microfluidic technology; during packaging, the aqueous phase was the mRNA solution (50 mM sodium acetate buffer solution, pH 4.0), and the ethanol phase was the lipid mixture (prepared by protonatable phospholipid, distearoyl phosphatidylcholine, cholesterol, and PEG-modified phospholipid in a molar ratio of 50:10:38.5:1.5), the total flow rate of the aqueous phase and the ethanol phase was 12 ml / min, and the volume ratio of the aqueous phase to the ethanol phase was 3:1; finally, the buffer of the packaged mRNA vaccine was replaced with PBS using a dialysis bag, and the Quan-iT RiboGreen RNA Assay Kit from Thermo Fisher was used to measure the concentrations of encapsulated and free mRNA, and calculate the mRNA packaging efficiency. The results showed that the packaging efficiency met the standards of the mRNA vaccine; after that, the encapsulated mRNA was stored at 4°C for later use.

[0196] Example 2: Detection of antibody expression and isolation and purification

[0197] In this example, according to the construction methods of the heavy chain, light chain sequences of antibodies and their expression plasmids disclosed in the literature, expression plasmids of the heavy and light chains of detection antibodies Z5 (Wang, Q.H. et al. Sci Transl Med 8, (2016)), Z6 (Dai, L. et al. Nat Immunol 22, 958-968, (2021)), 4G2 (Dai, L.P. et al. Nat Immunol 22, 958-968, (2021)), MZ24 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), Ab513 (Robinson, L.N. et al. Cell 162, 493-504, (2015)), 1F4 (Fibriansah, G. et al. Embo Mol Med 6, 358-371, (2014)), 3H5 (Renner, M. et al. Nature Immunology 19, 1248-56, (2018)), MZ-1 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), MZ-4 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), 5J7 (Fibriansah, G. et al. Embo Journal 31, 767-779, (2012)) and 5H2 (Cockburn, J.J.B. et al. Embo Journal 31, 767-779, (2012)) against different epitopes of the dengue virus prME protein were constructed respectively.

[0198] Among them, antibodies Z5, Z6 and 4G2 recognize the FL epitopes of Zika and dengue viruses, and the remaining antibodies (MZ-1, MZ-4, MZ24, Ab513, 1F4, 3H5, 5J7, and 5H2) are all high neutralizing activity antibodies that can recognize non-FL epitopes.

[0199] Antibody Expression

[0200] Fourteen to sixteen hours before transfection, plate the 293T cells with a relatively high density (for example, passage a 10-cm culture dish fully covered with 293T cells at a ratio of 1:3). Fourteen to sixteen hours later, when the cell density reaches over 70%, transfection can be carried out. During transfection, co-transfect the 293T cells with the heavy-chain and light-chain expression plasmids of the antibody constructed as described above at a ratio of 2:3. Four to six hours after transfection, wash the cells twice with PBS and then change to serum-free DMEM medium for continued culture. Collect the cell supernatant on the 3rd and 7th days after transfection respectively, centrifuge to remove cell debris, and mix the antibody supernatants obtained twice for subsequent antibody protein purification.

[0201] Antibody Purification

[0202] Connect a Protein A (5 ml) HP affinity column (purchased from GE) to an AKTA Purifier / Explorer / FPLC / START (GE). The operation process on the instrument is as follows: First, flush out the 20% ethanol in the column with water, and then equilibrate the column with a buffer of 20 mM Na3PO4, pH 7.0. After the conductivity on the instrument stabilizes, inject the above antibody supernatant through a 10-ml loop for binding to Protein A at a flow rate of 2 ml / min. After the UV stabilizes, add approximately 0.8 ml of 1 M Tris pH 9.0 buffer to the subsequent collection tube (the collection volume is about 3.2 ml). Then, change the program to elute the antibody bound to the column with 100% 0.1 M Gly pH 3.0, collect the eluate, and then use the method of concentration and buffer exchange to replace the antibody buffer with PBS. The obtained antibody solution can be used directly or aliquoted and stored at -80°C for later use.

[0203] Example 3: Expression and purification of dengue and Zika virus E proteins

[0204] In this example, construct the E protein expression plasmids of dengue and Zika viruses according to the method for soluble expression of the dengue and Zika virus E protein dimer disclosed in the literature (Kudlacek, S.T. et al. Sci Adv 7, (2021)).

[0205] Specifically, the nucleic acid coding sequences encoding amino acids 1-395 of the E protein of DV1 (THSTI-TRC-DV1-08 strain, GenBank: OP310803.1), DV2 (New Guinea C strain, GenBank: KM204118.1), DV3 (YN02 strain, GenBank: KF824903), DV4 (Guangzhou B5 strain in China, GenBank: AF289029), and the nucleic acid coding sequence encoding amino acids 1-404 of the E protein of ZIKV-SMGC-1 (GenBank: KX266255) were optimized according to the codon preference of mammalian cells. The optimized nucleic acid coding sequences of the E protein fragments of the above viruses are shown in SEQ ID NO: 120-124 respectively. Then, a Kozak sequence and the coding sequence of a signal peptide (as shown in SEQ ID NO: 119, encoding a signal peptide with an amino acid sequence as shown in SEQ ID NO: 60) were added to the 5' end of the above nucleic acid coding sequence, and a His tag was added to the 3' end for protein isolation and purification. The gene synthesis was entrusted to GenScript Biotech Corporation. Then, the synthesized gene fragments were digested with EcoRI at the 5' end and XhoI at the 3' end and cloned into the pCAGGS vector to construct E protein expression plasmids pCAGGS-DV1-sE, pCAGGS-DV2-sE, pCAGGS-DV3-sE, pCAGGS-DV4-sE, and pCAGGS-ZV-sE. HEK293F cells were used for protein expression, and HisTrapTM excel affinity chromatography column and Superdex200 Increase 10 / 300GL gel filtration chromatography column were used for protein purification.

[0206] Select HEK293F cells with good growth status and inoculate them into a new SMM293-TII medium at a density of 1-1.5x10 6 cells / ml the day before transfection, and culture them in a constant temperature shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, adjust the cell density to 2.5x10 6cells / ml. Prepare the transfection reagent (taking 10 ml of cells as an example). Dilute 10 μg of plasmid with 150 mM NaCl to a total volume of 250 μl and mix gently; dilute Sinofection transfection reagent with 150 mM NaCl to a total volume of 250 μl and mix gently. After separately standing the diluted plasmid and transfection reagent still for about 5 min, mix them gently. The total volume is 500 μl, and then stand at room temperature for 10 min. Dropwise add the transfection solution into the cell culture medium, gently shake the culture flask while adding the drops, and return it to the shaker for continued culture after mixing evenly. Add 350 μl of SMS-293-SUPI feeding solution 24 h after transfection, and then add the feeding solution (350 μl) every 48 h. After 5 days of transfection, collect the supernatant for protein purification.

[0207] Collect the cell supernatant 5 days after transfection. After centrifuging at 8000 rpm at 4 °C for 1 h, filter the supernatant through a 0.22 μm filter membrane. His affinity chromatography purification: First, wash the HisTrap TM excel affinity chromatography column with water filtered through a 0.22 μm filter membrane, and wash for about 3 - 5 column volumes. Then, wash the chromatography column with the HisTrap affinity chromatography equilibration buffer until the UV and conductivity detection lines are stable, indicating that the chromatography column has been equilibrated. Then, flow the cell supernatant through the chromatography column at a flow rate of 1 - 2 mL / min to bind the protein to the chromatography column. After all the supernatant has flowed through the chromatography column, wash the chromatography column with 5 - 10 column volumes of the affinity chromatography equilibration buffer until the UV detection line is stable. Finally, wash the chromatography column successively with elution buffers containing 50 mM, 300 mM, and 1 M imidazole, and identify the collected protein sample by SDS-PAGE. Further purify it by Superdex 200 Increase 10 / 300 GL gel filtration chromatography. Concentrate the sample obtained by affinity chromatography to less than 800 μl using a 10 kDa ultrafiltration concentrator tube, and transfer it to a 1.5 mL centrifuge tube. After centrifuging at 12000 g at 4 °C for 20 min, without sucking the precipitate, transfer the sample to a new 1.5 mL centrifuge tube. Centrifuge at 12000 g at 4 °C for 20 min to remove the air bubbles in the sample and prepare for loading. Equilibrate the Superdex 200 Increase 10 / 300 GL gel filtration chromatography column with the gel filtration chromatography equilibration buffer at a maximum flow rate of 0.5 mL / min until the UV detection line is stable, and load the sample using a 1 mL loop. After loading, wash the chromatography column with 20 / 50 equilibration buffer (20 mM Tris, 50 mM NaCl) at a flow rate of 0.5 mL / min, set the peak collection, and collect the sample at the elution peak. Identify the collected sample by SDS-PAGE. Concentrate the sample with correct expression identified by SDS-PAGE using a 10 kDa ultrafiltration concentrator tube and aliquot and store it in a -80 °C refrigerator for future use.

[0208] Example 4: Detection of Antigen Expression of Dengue Virus mRNA Vaccine

[0209] In this example, a series of dengue virus prME mRNA vaccines prepared in Example 1 were transfected into 293T cells, and then the FL epitope antibody and other neutralizing epitope antibodies prepared in Example 2 were used to detect the epitope situation and protein expression level of each prME.

[0210] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS. Before transfection, the cell density was made to reach over 70%, and the HEK293T cells were transfected with the mRNA vaccine constructed in Example 1. After 12 hours of transfection, the cell culture supernatant was discarded, the cells were digested with trypsin and collected. After washing the cells with PBS, the cell fixation and permeabilization solution (BD Cytofix / Cytoperm TM Fixation / Permeabilization Kit) was added, and the cells were incubated on ice for 20 minutes. After washing the cells twice with 1× washing solution (BD Cytofix / Cytoperm TM Fixation / Permeabilization Kit), the antibodies Z5 and Z6 that can recognize different epitopes of the dengue prME protein in Example 2 were added, and the cells were incubated in the dark on ice for 30 minutes. After washing the cells twice with 1× washing solution, FITC-labeled goat anti-human secondary antibody (Abcam, USA) was added, and the cells were incubated in the dark on ice for 30 minutes. After washing the cells twice with 1× washing solution, the cells were resuspended with PBS, and the cell fluorescence was detected on a BD FACSAria III flow cytometer. The data was analyzed using FlowJo 7.6.1.

[0211] The results are as Figures 1-4 shown. Figures 1-4 The results showed that:

[0212] For the DV1 mRNA vaccine, the FL epitope antibodies (Z5 and Z6) could bind to DV1-WT but not to the two mutants (DV1-EM3 and DV1-EM3-1), indicating that the FL epitope had been disrupted in the two mutants; the other neutralizing epitope antibodies (MZ24, Ab513, and 1F4) could bind to both DV1-WT and the two mutants, indicating that the other neutralizing epitopes had not changed in the two mutants, and the protein expression levels of the two mutants were similar to that of the wild type;

[0213] For the DV2 mRNA vaccine, the FL epitope antibodies (Z5, Z6, and 4G2) bind to DV2-WT but not to the two mutants DV2-EM3 and DV2-EM3-1, indicating that in the two mutants, the FL epitope has been disrupted; other neutralizing epitope antibodies (3H5, MZ-1, and MZ-4) can bind to both DV2-WT and the two mutants, indicating that other neutralizing epitopes have not changed in the two mutants, and the protein expression levels of the two mutants are similar to that of the wild type;

[0214] For the DV3 mRNA vaccine, the FL epitope antibodies (Z5 and Z6) bind to DV3-WT but not to the two mutants DV3-EM3 and DV3-EM3-1, indicating that in the two mutants, the FL epitope has been disrupted; other neutralizing epitope antibodies (MZ24, Ab513, and 5J7) can bind to both DV3-WT and the two mutants, indicating that other neutralizing epitopes have not changed in the two mutants, and the protein expression levels of the two mutants are similar to that of the wild type;

[0215] For the DV4 mRNA vaccine, the FL epitope antibodies (Z5 and Z6) bind to DV4-WT but not to the two mutants DV4-EM3 and DV4-EM3-1, indicating that in the two mutants, the FL epitope has been disrupted; other neutralizing epitope antibodies (MZ24, Ab513, and 5H2) can bind to both DV4-WT and the two mutants, indicating that other neutralizing epitopes have not changed in the two mutants, and the protein expression levels of the two mutants are similar to that of the wild type.

[0216] The above results indicate that in all of the above mutants, the FL epitope is disrupted, while other neutralizing antibody epitopes remain unchanged, and the protein expression levels of the mutants are comparable to or higher than that of the wild type. That is, the FL epitope in the recombinant antigen of the present application is disrupted, which avoids the production of FL epitope antibodies that cause ADE, thereby significantly reducing or eliminating the ADE effect; at the same time, the recombinant antigen can bind normally to other neutralizing epitope antibodies, indicating that it has the correct conformation of the E protein; and the recombinant antigen has a protein expression level similar to or higher than that of the wild type.

[0217] Example 5: Mouse Immunization Experiment of Dengue Virus mRNA Vaccine

[0218] In this example, female 6-8-week-old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the vaccine immunization experiment. The experiment was divided into an mRNA vaccine immunization group and a negative control group (i.e., the Sham group), with 6 mice in each group. For each immunization group, placebo (normal saline, as the Sham group) or the dengue mRNA vaccine prepared in Example 1 was intramuscularly injected on days 0 and 14, with a dose of 12 μg / mouse each time. Blood was collected 4 weeks after immunization, and the serum was separated at 4°C and inactivated at 56°C for 30 minutes, and then stored at -80°C for later use.

[0219] Example 6: Detection of specific antibody titers in the sera of immunized mice by enzyme-linked immunosorbent assay (ELISA)

[0220] In this example, the specific antibody titers against the antigen E protein in the sera of the immunized mice obtained in Example 5 were detected by ELISA.

[0221] Specifically, the following procedure was adopted:

[0222] (1) The dengue virus or Zika virus E protein prepared in Example 3 was diluted to 2 μg / ml with ELISA coating buffer (Solarbio, C1050). 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590) and placed at 4°C for 12 hours.

[0223] (2) The coating buffer was poured out, and PBS was added and washed once. 5% skim milk prepared with PBS was used as the blocking solution and added to the 96-well plate, 100 μl per well, and placed at room temperature for 1 hour for blocking. After blocking, it was washed once with PBS solution.

[0224] (3) During the blocking in step (2), the mouse serum samples were diluted with the blocking solution, starting from 10-fold and diluted in a 2-fold gradient. Then, 100 μl of the immunized serum dilution was added to each well of the ELISA plate, and the negative control was the addition of the blocking solution. It was incubated at 37°C for 2 hours, and then washed 4 times with PBST.

[0225] (4) Goat anti-mouse secondary antibody conjugated with HRP (Abcam, ab6789) diluted 1:2000 with the blocking solution was added, incubated at 37°C for 1 hour, and then washed 5 - 6 times with PBST. TMB chromogenic solution was added for color development. After reacting for an appropriate time, 2M hydrochloric acid was added to terminate the reaction, and the OD450 reading was detected on an enzyme-linked immunosorbent assay reader.

[0226] The antibody titer value was defined as the highest dilution factor of the serum with a reaction value greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) was still less than 2.1 times the background value, the titer of this sample was defined as half of the lowest dilution factor, i.e., 1:5.

[0227] The results of the ELISA experiment are as follows Figure 5 shown Figure 5 as follows: DV1-EM3 can induce DV1 virus E protein-specific antibody titers similar to those of DV1-WT; DV2-EM3-1, DV4-EM3, and DV4-EM3-1 vaccines also induced antibody titers similar to those of the corresponding wild-type vaccines; compared with DV3-WT, although the DV3 virus E protein-specific antibody titers induced by DV3-EM3 and DV3-EM3-1 were reduced, their antibody titer values were still relatively high (>10,000).

[0228] The above results indicate that the recombinant antigen of the present application can induce high levels of specific binding antibodies in mice, suggesting that it has high immunogenicity and can induce high levels of immune protection effects.

[0229] Example 7: Detection of neutralizing antibody titers in the sera of immunized mice by dengue virus micro-neutralization assay

[0230] In this example, the neutralizing antibody titers of the sera of mice immunized with each mRNA vaccine in Example 5 against the dengue virus of the corresponding serotype were quantitatively determined by the dengue virus micro-neutralization assay. The specific operation steps are as follows:

[0231] One day in advance, Vero cells were inoculated into 24-well cell culture dishes. When the cells grew to a confluence of more than 70% the next day, they were used for the neutralization test. The serum was serially diluted with DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106), and the virus was also diluted with DMEM medium containing 1% FBS. 200 μl of the serum and the virus solution were mixed in equal volumes. At the same time, the diluted virus was mixed with an equal volume of blank medium as a positive control and incubated at 37°C for 30 min. After incubation, the supernatant medium in the VERO cell plate was removed, and the above mixture was added to the cells, 350 μl / well. After incubating in a 37°C incubator for 1 hour, 150 μl of DMEM medium containing 1% FBS was added to each well, and then continued to culture in a 37°C incubator for 3 days. After 3 days, the cell culture plate was taken out, all the supernatant was discarded, and the cells were washed once with PBS. Then, 150 μL of trypsin was added to each well to digest the cells, and the cells were left standing in a 37°C constant temperature incubator for 5 minutes. Then, 150 μL of PBS containing 10% FBS was added to the wells to terminate the trypsin digestion. The digested cells were dispersed and mixed evenly. All the cells in the 24-well plate were transferred to a 96-well pointed bottom plate, and the cells were collected by centrifugation and then washed once with PBS. Then, 100 μL of Fixation and Permeabilization solution (BD, 554722) was added to each well of the 96-well round bottom plate, and the cells were fixed in the dark at 4°C for 30 minutes, and then the cells were collected by centrifugation at 800g for 5 minutes and washed twice with 1×Perm / Wash buffer (BD, 554723). AF-488-labeled Z6 antibody (Z6-AF488) was added, 100 μl per well, and the cells were stained in the dark at 4°C for 30 minutes. After staining, the cells were collected by centrifugation and washed twice with 1×Perm / Wash buffer. Finally, the cells were resuspended with 200 μL of PBS, transferred to a flow tube, and the FITC fluorescence positive rate of the cells was detected with FACS Canto II. GraphPad Prism software was used to perform nonlinear fitting on the data to calculate the serum dilution factor corresponding to neutralizing 50% of the cell infections, which was the neutralization titer value (IC50). When the serum at the lowest dilution factor still could not neutralize 50% of the cell infections, the IC50 of this sample was defined as half of the lowest dilution factor.

[0232] The results are as Figure 6 shown Figure 6 and show that compared with the corresponding WT vaccines, the DV1-EM3, DV2-EM3-1, DV3-EM3, and DV4-EM3 vaccines all induced comparable virus neutralizing antibody titers, and the virus neutralizing antibody titers induced by DV3-EM3-1 and DV4-EM3-1 were slightly lower. In particular, compared with DV3-WT, the DV3-EM3 vaccine induced fewer binding antibodies (Figure 5 ) but the neutralizing activity of the vaccine serum did not decrease. Figure 6 ) indicating that the DV3-EM3 vaccine induced a higher proportion of antibodies with high neutralizing activity.

[0233] The above results indicate that the recombinant antigen of the present application can induce a high level of neutralizing antibody titer in mice, suggesting its high immunogenicity, and thus can induce a high level of immune protection effect.

[0234] Example 8: Determination of cross-antibody titer

[0235] Since the FL epitope is highly conserved in dengue virus and Zika virus, the FL epitope antibody has broad-spectrum properties, that is, such antibodies can cross-bind the E proteins of all serotypes of dengue virus and Zika virus. However, such antibodies generally have poor virus neutralizing activity and are prone to inducing the ADE effect. The FL epitope antibodies induced by any serotype of dengue virus vaccine can cross-bind the E proteins of the other three serotypes of dengue virus and Zika virus. Therefore, evaluating the cross-antibody titer of the vaccine can reflect the amount of FL epitope antibodies induced by the vaccine and the ADE risk of the vaccine. The cross-antibody titer was determined by ELISA method, and the specific method was referred to Example 6.

[0236] The determination results of the cross-antibody titers induced by each mRNA vaccine immunization in Example 5 are as Figures 7-10 shown Figures 7-10 showing:

[0237] For the DV1 vaccine, a large number of antibodies induced by DV1-WT can cross-bind the E proteins of DV2 / 3 / 4 and ZIKV viruses, while the cross-antibody titer of DV1-EM3 was significantly reduced, and cross-binding antibodies could not even be detected in some mice. It can be speculated that the ADE risk of the DV1-EM3 vaccine against DV2 / 3 / 4 and ZIKV viruses is significantly reduced;

[0238] Similar results were also obtained for the DV2 / 3 / 4 vaccines, that is, compared with the corresponding WT vaccines, the cross-antibody titers induced by each mutant vaccine were significantly decreased, and their ADE risks were also significantly reduced.

[0239] The above results indicate that the cross-antibodies induced by the recombinant antigen of the present application against the E proteins of other serotypes of DV or ZIKV viruses are significantly reduced, thereby greatly reducing the risk of the vaccine-induced ADE effect and having high safety.

[0240] Example 9: Determination of serum neutralizing antibody titer and cross-antibody titer of DV1-EM3-1 and DV2-EM3 vaccines

[0241] In this example, the neutralizing antibody titers and cross - antibody titers of the sera of mice immunized with DV1 - EM3 - 1 and DV2 - EM3 vaccines obtained in Example 5 were measured. The method for measuring the neutralizing antibody titer referred to Example 7, and the method for measuring the cross - antibody titer referred to Example 8.

[0242] The results of the serum neutralizing antibody titer measurement are as Figure 11 shown, Figure 11 showing that compared with the corresponding WT vaccine, DV1 - EM3 - 1 induced comparable virus - neutralizing antibody titers, and although the neutralizing antibody titers induced by DV2 - EM3 decreased, they were still above 1,000, all showing good immunogenicity.

[0243] The results of the cross - antibody titer measurement are as Figures 12-13 shown, Figures 12-13 showing that compared with the WT vaccine, the cross - antibody titers of DV1 - EM3 - 1 and DV2 - EM3 vaccines against heterologous serotype dengue viruses and Zika virus were significantly and substantially reduced. It is thus speculated that the ADE risks against heterologous serotype dengue viruses and Zika virus are also substantially reduced.

[0244] Example 10: Evaluation of the in vitro ADE effect induced by the vaccine

[0245] In this example, female 6 - 8 - week - old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the vaccine immunization experiment. The experiment was divided into an mRNA vaccine immunization group and a negative control group (i.e., the Sham group), with 8 mice in each group. For each immunization group, a placebo (normal saline, as the Sham group) or the DV1 - WT, DV2 - WT, DV3 - WT, DV4 - WT, DV1 - EM3 - 1, DV2 - EM3 - 1, DV3 - EM3, DV4 - EM3 monovalent dengue mRNA vaccines prepared in Example 1 were intramuscularly injected on days 0 and 21, respectively. The injection dose each time was 1 μg / mouse. Blood was collected 2 weeks after the booster immunization, and the serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at - 80°C for later use.

[0246] Using the K562 cell model (ATCC, catalog number CCL - 243) with high expression of the FcγRIIA receptor, the ADE effects of the sera of BALB / c mice immunized with the above - mentioned monovalent mRNA vaccines against the other three serotype viruses were measured, such as the ADE effects of DV1 - WT, DV1 - EM3, and DV1 - EM3 - 1 immune sera against DENV2, 3, and 4 viruses. The specific operation steps are as follows:

[0247] Take out the vaccine immune serum and melt it at 4°C. Gently flick to mix evenly and centrifuge briefly in a centrifuge. In a laminar flow hood, serially dilute the mouse serum in a 96-well U-bottom plate (Corning 3799) at a 4-fold ratio with RPMI 1640 medium containing 1% fetal bovine serum (Invitrogen, C11875500BT). Dilute the DENV virus to 2×10 6 FFU / mL with RPMI 1640 medium containing 1% fetal bovine serum. Mix the serum dilutions with the 3 DENV viruses in equal volumes and incubate at room temperature for 20 minutes. Centrifuge the cultured K562 cells at 800 g for 5 minutes, resuspend them with RPMI-1640 medium containing 1% FBS, count the cells, and then adjust the cell density to 4×10 6 / mL. Subsequently, add 10 μL per well to the virus-serum mixture and incubate in a 37°C cell culture incubator for 1 hour. Supplement with RPMI1640 medium containing 1% FBS, 100 μL per well, and continue culturing in a 37°C cell culture incubator for 12 - 14 hours.

[0248] After 12 hours, transfer the cells to a 96-well plate, centrifuge at 800 g for 5 minutes, remove the supernatant, then wash once with PBS and centrifuge to collect the cells. Add 100 μL per well of Fixation and Permeabilization solution (BD554722) cell fixative to the 96-well U-bottom plate and incubate in a 4°C refrigerator for 30 min. Centrifuge at 800 g for 5 minutes, collect the cells and wash twice with 1×Perm / Wash buffer (BD 554723). Add Z6 antibody labeled with AF488 (Z6-AF488), 100 μL per well, and incubate in a 4°C refrigerator for 30 minutes; centrifuge at 800 g for 3 minutes, collect the cells, and wash twice with 1×Perm / Wash buffer. Resuspend the cells with 100 μL per well of PBS and use a flow cytometer to detect the proportion of positive cells infected with the virus.

[0249] The results are as Figures 14-17As shown, it shows that DENV virus does not infect K562 cells after being mixed with Sham immune serum; immune sera of four wild-type vaccines (DV1-WT, DV2-WT, DV3-WT, and DV4-WT) can detect strong in vitro ADE effects at different dilutions, that is, WT vaccine sera can enhance the infection of dengue virus to K562 cells, and the cell infection rate increases; compared with the above wild-type vaccines, the in vitro ADE effects induced by mutant vaccine (DV1-EM3, DV2-EM3, DV3-EM3, DV4-EM3, DV1-EM3-1, DV2-EM3-1, DV3-EM3-1, and DV4-EM3-1) mouse sera are significantly and substantially reduced, which indicates that the risk of mutant antigens inducing ADE effects is significantly and substantially reduced.

[0250] Example 11: Evaluation of in vivo ADE effects induced by vaccines

[0251] In this example, the immune sera of BALB / c mice immunized with vaccines DV4-WT, DV4-EM3, and DV4-EM3-1 obtained in Example 10 were mixed with DENV2 virus and then subcutaneously injected into AG129 mice to detect the in vivo ADE effects of the immune sera of DENV4 vaccines induced on DENV2 virus in the AG129 mouse model.

[0252] Using AG129 mice as the infection model, BALB / c mice were immunized with different vaccines or PBS. Four weeks after the booster immunization, blood was collected and serum was separated, heat-inactivated, and the sera of different mice in each vaccine group were mixed together, aliquoted, and stored at -80 °C until use. Before adoptive transfer to AG129 mice, the serum was first diluted 1000-fold with PBS to 50 μL, and then mixed with an equal volume of DENV2 virus (6000 FFU), and the virus-serum mixture was subcutaneously injected into AG129 mice. The survival rate and weight loss of the mice were monitored daily, and mice with an initial weight loss of 25% were euthanized.

[0253] The results are as Figure 18 shown, which shows that compared with adoptive transfer of PBS immune serum, the immune serum of wild-type vaccine DV4-WT induced an earlier death time of AG129 mice, showing a significant ADE effect; while the immune sera of mutant vaccines DV4-EM3 and DV4-EM3-1 did not cause faster death of AG129 mice and had no ADE effect.

[0254] Example 12: Immunization of mice with tetravalent dengue virus mRNA vaccine

[0255] In this example, female 6-8-week-old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the tetravalent dengue virus vaccine immunization experiment. The experiment was divided into a negative control group (Sham group), a tetravalent wild-type vaccine group (tWT group), and a tetravalent mutant vaccine group (tMut group), with 8 mice in each group. Among them, the negative control group was immunized with normal saline. The tWT vaccine was a mixture of four vaccines, DV1-WT, DV2-WT, DV3-WT, and DV4-WT, prepared in Example 1, with 3 μg / dose of each vaccine, that is, a total of 12 μg / dose. The tMut vaccine was a mixture of four vaccines, DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, prepared in Example 1, with 3 μg / dose of each vaccine, that is, a total of 12 μg / dose. For each immunized group, intramuscular injection was performed on day 0 and day 21 respectively, and blood was collected 6 weeks after the primary immunization. The serum was separated at 4°C and inactivated at 56°C for 30 minutes and then stored at -80°C for later use.

[0256] Example 13: Expression and purification of FL antibody scfv

[0257] In this example, according to the heavy and light chain sequences of antibodies disclosed in the literature, the dengue virus FL antibodies Z5-scfv (Wang, Q.H. et al. Sci Transl Med 8, (2016)) and Z6-scfv (Dai, L. et al. Nat Immunol 22, 958-968, (2021)) were constructed respectively. After adding a signal peptide and a His tag to the 5' and 3' ends of the scfv antibody amino acid sequence, optimization was carried out according to the mammalian cell codon preference to obtain the nucleic acid coding sequences of Z5-scfv and Z6-scfv antibodies, as shown in SEQ ID NO:125 and SEQ ID NO:126 respectively. Then, a Kozak sequence and a stop codon were added to the 5' and 3' ends of the above nucleic acid coding sequences, and then entrusted to GenScript Biotech Corporation for gene synthesis. The synthesized gene fragments were cloned into the pCAGGS vector by double digestion with 5'EcoRI and 3'XhoI to construct the E protein expression plasmids pCAGGS-Z5-scfv and pCAGGS-Z6-scfv. HEK293F cells were used for protein expression, and HisTrapTM excel affinity chromatography column and Superdex200 Increase 10 / 300GL gel filtration chromatography column were used for protein purification.

[0258] Select HEK293F cells with good growth status. One day before transfection, at a density of 1-1.5x10 6Inoculate into a new SMM293-TII medium at a density of cells / ml and culture in a constant temperature shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, adjust the cell density to 2.5×10 6 cells / ml. Prepare the transfection reagent (taking 10 ml of cells as an example), dilute 10 μg of plasmid with 150 mM NaCl to a total volume of 250 μl, and gently mix; dilute Sinofection transfection reagent with 150 mM NaCl to a total volume of 250 μl, and gently mix; after separately standing the diluted plasmid and transfection reagent gently for about 5 min, mix gently, with a total volume of 500 μl, and then stand at room temperature for 10 min. Add the transfection solution dropwise into the cell culture medium, gently shake the culture flask while dropping, and after shaking evenly, put it back into the shaker for continued culture. Add 350 μl of SMS-293-SUPI feeding solution 24 h after transfection, and then add the feeding solution (350 μl) every 48 h thereafter. Five days after transfection, collect the supernatant for protein purification.

[0259] Collect the cell supernatant 5 days after transfection, centrifuge at 8000 rpm and 4°C for 1 h, and then filter the supernatant through a 0.22 μm filter membrane. His affinity chromatography purification: First, rinse HisTrap with water that has been filtered through a 0.22 μm filter membrane TMThe Excel affinity chromatography column was rinsed with approximately 3 - 5 column volumes. Subsequently, it was rinsed with the HisTrap affinity chromatography equilibration buffer until the UV and conductivity detection lines were stable, indicating that the chromatography column was equilibrated. Then, the cell supernatant was passed through the chromatography column at a flow rate of 1 - 2 mL / min to bind the protein to the column. After all the supernatant had passed through the column, the column was rinsed with 5 - 10 column volumes of the affinity chromatography equilibration buffer until the UV detection line was stable. Finally, the column was rinsed successively with elution buffers containing 50 mM, 300 mM, and 1 M imidazole. The collected protein samples were prepared for SDS - PAGE identification. Further purification was performed using Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The sample obtained from affinity chromatography was concentrated to less than 800 μl using a 10 kDa ultrafiltration concentrator tube and then transferred to a 1.5 mL centrifuge tube. After centrifugation at 12000 g for 20 min at 4°C, the sample was transferred to a new 1.5 mL centrifuge tube ensuring no precipitate was aspirated. After centrifugation at 12000 g for 20 min at 4°C to remove air bubbles in the sample, it was ready for loading. The Superdex 200 Increase 10 / 300 GL gel filtration chromatography column was equilibrated with the gel filtration chromatography equilibration buffer at a maximum flow rate of 0.5 mL / min until the UV detection line was stable, and then loaded using a 1 mL loop. After loading, the column was rinsed with 20 / 50 equilibration buffer (20 mM Tris, 50 mM NaCl) at a flow rate of 0.5 mL / min, and peak collection was set to collect the samples at the elution peaks. The collected samples were prepared for SDS - PAGE identification. The samples with correct expression identified by SDS - PAGE were concentrated using a 10 kDa ultrafiltration concentrator tube, aliquoted, and stored at - 80°C in the refrigerator for future use.

[0260] Example 14: Detection of antibodies against the FL epitope induced by the tetravalent dengue mRNA vaccine by competitive enzyme - linked immunosorbent assay (ELISA)

[0261] In this example, through a competitive ELISA experiment, the serum of mice immunized with the tetravalent dengue mRNA vaccine obtained in Example 12 was used to detect the antibody level against the FL epitope of the antigen E protein induced by the tetravalent dengue vaccine.

[0262] Specifically, the following procedure was adopted:

[0263] (1) The dengue virus E protein prepared in Example 3 was diluted to 2 μg / mL with the ELISA coating buffer (Solarbio, C1050). 100 μL was added to each well of a 96 - well ELISA plate (Coring, 3590) and placed at 4°C for 12 hours.

[0264] (2) Discard the coating solution, add PBS, and wash once; Add 5% skim milk prepared with PBS as the blocking solution to the 96-well plate, 100 μL per well, and place at room temperature for 1 hour for blocking; After blocking, wash once with PBS solution;

[0265] (3) During the blocking in step (2), dilute the scFv competitive antibody mixture (Z5-scfv and Z6-scfv mixed at a concentration ratio of 1:1) with the blocking solution to a concentration of 20 μg / ml. In the non-competitive ELISA assay, add 50 μL of blank blocking solution. In the competitive ELISA assay, add 50 μL of scFv competitive antibody. The negative control is to add blank blocking solution, and incubate at 37 °C for 1 hour;

[0266] (4) During the blocking in step (3), dilute the mouse immune serum sample with the blocking solution. The serum sample starts from 10,000-fold and is diluted in a 2-fold gradient; Then, add 50 μL of the diluted immune serum to each well in the ELISA plate. The negative control is to add the blocking solution, incubate at 37 °C for 2 hours, and then wash 4 times with PBST;

[0267] (5) Add goat anti-mouse secondary antibody conjugated with HRP (Abcam, ab6789) diluted 1:2000 with the blocking solution, incubate at 37 °C for 1 hour, and then wash 5 - 6 times with PBST; Add TMB chromogenic solution for color development. After reacting for an appropriate time, add 2 M hydrochloric acid to terminate the reaction, and detect the OD450 reading on the microplate reader.

[0268] The antibody titer value is defined as the highest dilution factor of the serum when the reaction value is greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.1 times the background value, the titer of this sample is defined as half of the lowest dilution factor, that is, 1:5.

[0269] The results of the competitive ELISA experiment are as Figure 19 shown Figure 19Show: When no FL epitope scfv competitive antibody was added (tWT and tMut), all E protein-specific antibodies in mouse sera could bind to the coated E protein. When the FL epitope scfv competitive antibody was added (tWT-C and tMut-C), the FL epitope antibodies in mouse sera could not bind to the coated E protein, while the antibodies of the remaining epitopes could bind to the coated E protein normally. Therefore, the difference between the two could represent the number of FL epitope antibodies in mouse sera. After adding the FL epitope scfv competitive antibody, the serum antibody titer of the tWT vaccine decreased significantly, indicating that the tWT vaccine induced a large number of FL epitope antibodies. In comparison, the antibody titers measured for the tMut vaccine serum remained unchanged whether the FL epitope scfv competitive antibody was added or not, indicating that the tMut vaccine did not induce FL epitope antibodies. The above results indicate that the mutant antigen of the present application does not induce FL antibodies with high ADE activity in mice, suggesting a low ADE risk.

[0270] Example 15: Detection of neutralizing antibodies induced by tetravalent dengue vaccine

[0271] Through the dengue virus micro-neutralization assay, the neutralizing antibody titers against four serotypes of dengue virus in the sera of mice immunized with the tetravalent dengue mRNA vaccine obtained in Example 12 were quantitatively determined. The specific operation steps are as follows:

[0272] One day in advance, Vero cells were inoculated into 24-well cell culture dishes. When the cell confluence reached more than 70% the next day, they were used for the neutralization test. The serum was serially diluted with DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106), and the virus was also diluted with DMEM medium containing 1% FBS. 50 μL of the serum and the virus solution were mixed in equal volumes. At the same time, the diluted virus was mixed with an equal volume of blank medium as a positive control and incubated at 37 °C for 30 min. After incubation, the supernatant medium in the VERO cell plate was removed, and the above mixed solution was added to the cells, 100 μL / well, and continued to be cultured in an incubator at 37 °C for 3 days. After 3 days, the cell culture plate was taken out, all the supernatant was discarded, and the cells were washed once with PBS. The medium was removed, and the cells were washed once with PBS. 50 μl of fixation and permeabilization solution (BD Cytofix / Cytoperm Soln Kit 554714) was added to each well and fixed at 4 °C for 30 min. The cells were washed twice with PBST, 50 μL of MZ24-HRP dilution solution (concentration 2 μg / ml) was added to each well, and incubated at 4 °C for 1 h. The cells were washed three times with PBST, 50 μL of TMB chromogenic solution was added to each well, terminated with 2 M sulfuric acid after 8 min, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer). The data was non-linearly fitted using GraphPad Prism software to calculate the serum dilution factor corresponding to neutralizing 50% of the cell infections, which was the neutralization titer value (NT 50 ). When the serum at the lowest dilution factor still could not neutralize 50% of the cell infections, the NT 50 of the sample was defined as half of the lowest dilution factor.

[0273] The results were as Figure 20 shown, which showed that compared with the negative control group, both the tWT and tMut vaccines induced high-titer neutralizing antibody immune responses against the four serotype viruses. In particular, the tMut vaccine also induced a balanced neutralizing antibody response against the four serotype viruses.

[0274] Example 16: Evaluation of the protective effect of a tetravalent dengue vaccine against virus challenge

[0275] In this example, an AG129 mouse lethal model was used to detect the protective effect of the tetravalent dengue mRNA vaccine against virus challenge.

[0276] Specifically, 6- to 8-week-old AG129 mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were used for the immunization and challenge experiments of the tetravalent dengue virus vaccine. The experiments were divided into a negative control group (Sham group) and a tetravalent mutant vaccine group (tMut group), with 12 mice in each group. Among them, the negative control group was immunized with normal saline, and the tMut vaccine was composed of four vaccines, DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, prepared in Example 1. Each vaccine was 3 μg / dose, that is, a total of 12 μg / dose. For each immunization group, intramuscular injection was performed on days 0 and 21, and on the 49th day after the primary immunization, the mice in each group were randomly and evenly distributed. Half of them were subcutaneously injected with 1×10^6 FFU DENV1 virus, and the other half were subcutaneously injected with 1×10^6 FFU DENV2 virus. After the DENV virus challenge, the animals were weighed daily and monitored for clinical signs of the disease. Mice with an initial weight loss of 25% were euthanized due to animal welfare, and the mortality rate was finally counted.

[0277] The results are as Figure 21 shown, which shows that after the DENV1 and DENV2 virus challenges, all the mice in the Sham group died, while all the mice in the tMut vaccine group survived, indicating that the tMut vaccine can provide excellent immune protection for AG129 mice.

[0278] Example 17: Preparation of Dengue Virus mRNA Vaccine

[0279] In this example, a series of mRNA constructs encoding the full-length dengue virus prME protein (i.e., including the prM protein + E protein) were designed, including:

[0280] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein shown in SEQ ID NO:1;

[0281] DV1-EM1, encoding the prME antigen protein shown in SEQ ID NO:2;

[0282] DV1-EM2, encoding the prME antigen protein shown in SEQ ID NO:5;

[0283] DV1-EM4, encoding the prME antigen protein shown in SEQ ID NO:6;

[0284] DV1-EM5, encoding the prME antigen protein shown in SEQ ID NO:7;

[0285] DV1-EM6, encoding the prME antigen protein shown in SEQ ID NO:8;

[0286] DV1-EM7 encodes the prME antigen protein as shown in SEQ ID NO:9;

[0287] DV1-EM8 encodes the prME antigen protein as shown in SEQ ID NO:10;

[0288] DV1-EM9 encodes the prME antigen protein as shown in SEQ ID NO:11;

[0289] DV1-EM10 encodes the prME antigen protein as shown in SEQ ID NO:12;

[0290] DV1-EM11 encodes the prME antigen protein as shown in SEQ ID NO:13;

[0291] DV1-EM12 encodes the prME antigen protein as shown in SEQ ID NO:14;

[0292] DV2-WT (i.e., DV2 wild type) encodes the prME antigen protein as shown in SEQ ID NO:15;

[0293] DV2-EM1 encodes the prME antigen protein as shown in SEQ ID NO:16;

[0294] DV2-EM2 encodes the prME antigen protein as shown in SEQ ID NO:19;

[0295] DV2-EM4 encodes the prME antigen protein as shown in SEQ ID NO:20;

[0296] DV2-EM5 encodes the prME antigen protein as shown in SEQ ID NO:21;

[0297] DV2-EM6 encodes the prME antigen protein as shown in SEQ ID NO:22;

[0298] DV2-EM7 encodes the prME antigen protein as shown in SEQ ID NO:23;

[0299] DV2-EM8 encodes the prME antigen protein as shown in SEQ ID NO:24;

[0300] DV2-EM9 encodes the prME antigen protein as shown in SEQ ID NO:25;

[0301] DV2-EM10 encodes the prME antigen protein as shown in SEQ ID NO:26;

[0302] DV2-EM11 encodes the prME antigen protein as shown in SEQ ID NO:27;

[0303] DV2-EM12 encodes the prME antigen protein as shown in SEQ ID NO:28;

[0304] DV3-WT (i.e., DV3 wild type) encodes the prME antigen protein as shown in SEQ ID NO:29;

[0305] DV3-EM1 encodes the prME antigen protein as shown in SEQ ID NO:30;

[0306] DV3-EM2 encodes the prME antigen protein as shown in SEQ ID NO:33;

[0307] DV3-EM4 encodes the prME antigen protein as shown in SEQ ID NO:34;

[0308] DV3-EM5 encodes the prME antigen protein as shown in SEQ ID NO:35;

[0309] DV3-EM6 encodes the prME antigen protein as shown in SEQ ID NO:36;

[0310] DV3-EM7 encodes the prME antigen protein as shown in SEQ ID NO:37;

[0311] DV3-EM8 encodes the prME antigen protein as shown in SEQ ID NO:38;

[0312] DV3-EM9 encodes the prME antigen protein as shown in SEQ ID NO:39;

[0313] DV3-EM10 encodes the prME antigen protein as shown in SEQ ID NO:40;

[0314] DV3-EM11 encodes the prME antigen protein as shown in SEQ ID NO:41;

[0315] DV3-EM12 encodes the prME antigen protein as shown in SEQ ID NO:42;

[0316] DV4-WT (i.e., DV4 wild type) encodes the prME antigen protein as shown in SEQ ID NO:43;

[0317] DV4-EM1 encodes the prME antigen protein as shown in SEQ ID NO:44;

[0318] DV4-EM2 encodes the prME antigen protein as shown in SEQ ID NO: 47;

[0319] DV4-EM4 encodes the prME antigen protein as shown in SEQ ID NO: 48;

[0320] DV4-EM5 encodes the prME antigen protein as shown in SEQ ID NO: 49;

[0321] DV4-EM6 encodes the prME antigen protein as shown in SEQ ID NO: 50;

[0322] DV4-EM7 encodes the prME antigen protein as shown in SEQ ID NO: 51;

[0323] DV4-EM8 encodes the prME antigen protein as shown in SEQ ID NO: 52;

[0324] DV4-EM9 encodes the prME antigen protein as shown in SEQ ID NO: 53;

[0325] DV4-EM10 encodes the prME antigen protein as shown in SEQ ID NO: 54;

[0326] DV4-EM11 encodes the prME antigen protein as shown in SEQ ID NO: 55;

[0327] DV4-EM12 encodes the prME antigen protein as shown in SEQ ID NO: 56.

[0328] Next, according to the codon preference of mammalian cells, the nucleic acid sequence encoding the above-mentioned recombinant dengue virus prME antigen protein was optimized to obtain an optimized nucleic acid coding sequence. Among them, the optimized nucleic acid coding sequences of DV1-WT, DV2-WT, DV3-WT, and DV4-WT are shown in SEQ ID NO: 114-117 respectively. The optimized nucleic acid coding sequences of DV1-EM1, DV1-EM2, DV1-EM4, DV1-EM5, DV1-EM6, DV1-EM7, DV1-EM8, DV1-EM9, DV1-EM10, DV1-EM11, and DV1-EM12 are shown in SEQ ID NO: 62, 65-74 respectively. The optimized nucleic acid coding sequences of DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, and DV2-EM12 are shown in SEQ ID NO: 75, 78-87 respectively. The optimized nucleic acid coding sequences of DV3-EM1, DV3-EM2, DV3-EM4, DV3-EM5, DV3-EM6, DV3-EM7, DV3-EM8, DV3-EM9, DV3-EM10, DV3-EM11, and DV3-EM12 are shown in SEQ ID NO: 88, 91-100 respectively. The optimized nucleic acid coding sequences of DV4-EM1, DV4-EM2, DV4-EM4, DV4-EM5, DV4-EM6, DV4-EM7, DV4-EM8, DV4-EM9, DV4-EM10, DV4-EM11, and DV4-EM12 are shown in SEQ ID NO: 101, 104-113 respectively. Then, the nucleic acid coding sequence of the Kozak sequence and the signal peptide (such as SEQ ID NO: 118, whose encoded amino acid sequence is the signal peptide shown in SEQ ID NO: 58) was added to the 5' end of each nucleic acid coding sequence, and a stop codon was added to its 3' end. Then, GenScript Biotech Corporation was commissioned for gene synthesis, and the synthesized gene fragment was recombined onto the pHRNT vector (invention patent ZL202110224383.0) stored in the laboratory to obtain the template plasmid for preparing the mRNA vaccine. The preparation method of the mRNA vaccine refers to Example 1.

[0329] Example 18: Detection of Antigen Expression of Dengue Virus mRNA Vaccine

[0330] In this example, a series of dengue virus prME mRNA vaccines prepared in Example 17 were transfected into 293T cells, and then the FL epitope antibody and other neutralizing epitope antibodies prepared in Example 2 were used to detect the epitope situation and protein expression level in each prME. For the detailed experimental procedures, refer to Example 4. The cell fluorescence was detected on a BD FACSAria III flow cytometer, and the relative positive rate of each mutant vaccine was calculated with the positive cell rate of the WT vaccine being 100%.

[0331] The results are as Figures 22-25 shown, which show that

[0332] For the DV1 mRNA vaccine, the FL epitope antibodies (Z5 and Z6) could bind to DV1-WT, but the binding decreased or no binding was observed with all DV1 mutants, indicating that in all DV1 mutants, the FL epitope had been partially or completely disrupted; the neutralizing epitope antibody Ab513 could bind to both DV1-WT and all DV1 mutants, indicating that this neutralizing epitope still existed in all DV1 mutants, and all DV1 mutants had a relatively high protein expression level.

[0333] Similar results were obtained for the DV2 mRNA vaccine, DV3 mRNA vaccine, and DV4 mRNA vaccine.

[0334] Example 19: Evaluation of the protective effect of DV2 mutant mRNA vaccine against virus challenge

[0335] In this example, an AG129 mouse lethal model was used to detect the protective effect against virus challenge of different mutants of the DV2 mRNA vaccine (DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, DV2-EM12) prepared in Example 17.

[0336] Specifically, 6-8-week-old AG129 mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were used for vaccine immunization and challenge experiments. The experiments were divided into a negative control group (Sham group) and each mutant vaccine group (DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, DV2-EM12 groups), with 5 mice in each group. Among them, the negative control group was immunized with normal saline, and each dose of the mutant vaccine was 15 μg / mouse. For each immunization group, intramuscular injection was performed on the 0th day and the 21st day, and on the 49th day after the primary immunization, the mice were subcutaneously injected with 1×10^6 FFU DENV2 virus. After the DENV virus challenge, the animals were weighed daily and the clinical signs of the disease were monitored. Mice with an initial weight loss of 25% were euthanized due to animal welfare, and finally the mortality rate was counted.

[0337] The results are as Figure 26 shown, which show that after the DENV2 virus challenge, all the mice in the Sham group died, while all the mice in the DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, DV2-EM12 vaccine groups survived; these results indicate that each mutant vaccine can provide excellent immune protection for AG129 mice.

[0338] Example 20: Preparation of Dengue Virus-Like Particle Vaccine

[0339] For flavivirus, recombinant expression of the full-length prME protein can form virus-like particles. In this example, a series of mammalian cell expression plasmids encoding the full-length prME protein of dengue virus were designed, including:

[0340] DV1-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:4;

[0341] DV2-EM3-1, encoding the prME antigen protein as shown in SEQ ID NO:18;

[0342] DV3-EM3, encoding the prME antigen protein as shown in SEQ ID NO:31;

[0343] DV4-EM3, encoding the prME antigen protein as shown in SEQ ID NO:45;

[0344] Next, according to the codon preference of mammalian cells, the nucleic acid sequence encoding the above recombinant dengue virus prME antigen protein was optimized to obtain optimized nucleic acid coding sequences. The optimized nucleic acid coding sequences of DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3 are shown in SEQ ID NO:64, 77, 89, and 102 respectively. Then, the Kozak sequence and the nucleic acid coding sequence of the signal peptide (such as SEQ ID NO:118, encoding the signal peptide with the amino acid sequence shown in SEQ ID NO:58) were added to the 5' end of each nucleic acid coding sequence, and a stop codon was added to the 3' end. Then, GenScript Biotech Corporation was commissioned for gene synthesis, and the synthesized gene fragment was recombined onto the pCAGGS vector stored in the laboratory to obtain the template plasmid for preparing the mRNA vaccine. HEK293T cells were used to express virus-like particles. HiTrap Capto Q ImpRes anion exchange chromatography column and SuperoseTM6 Increase 10 / 300GL gel filtration chromatography column were used for the purification of virus-like particles. One day before transfection, the 293T cells in a 15-cm culture dish were passaged at a ratio of 1:3. After 14 - 16 h, when the cell density reached over 70%, transfection could be carried out. Before transfection, the cell culture medium was replaced with fresh antibiotic-free DMEM medium. Prepare the transfection system: Transfect 30 μg of plasmid into each 15-cm culture dish, add it to 500 μl of opti-MEM, mix gently and let stand for 5 min. Add 150 μl Transfection Reagent to 350 μl of opti-MEM, mix gently and let stand for 5 min. Then mix the two and incubate at room temperature for 15 min. 4) Mix the transfection system, slowly drop it into the antibiotic-free cell culture supernatant in a 15-cm culture dish, mix gently, and place it in an incubator at 37°C and 5% CO2 for culture. 5) After 4 - 6 h, discard the cell culture supernatant containing the transfection reagent, replace it with antibiotic-free and serum-free DMEM medium, and continue to culture for 48 h to harvest the cells and the expression supernatant.

[0345] Prepare a concentration cup and select a filter membrane with a molecular weight cut-off of 100 kDa according to the size of VLP. Collect the cell supernatant 2 days after transfection. After centrifuging at 3500 rpm and 4°C for 15 min, pour the supernatant into the concentration cup, slowly open the liquid nitrogen valve, and concentrate the supernatant to 5 - 10 ml. Change the buffer to 20 mM Tris / 150 mM NaCl, which contains 0.1 mM EDTA, pH 8.0.

[0346] Purification by HiTrap Capto Q ImpRes anion exchange chromatography column: First, rinse the HiTrap Capto Q ImpRes anion exchange chromatography column with water filtered through a 0.22 μm filter membrane for about 3 - 5 column volumes. Then, rinse the chromatography column with 20 mM Tris / 150 mM NaCl equilibration buffer until the UV and conductivity detection lines are stable, indicating that the chromatography column has been equilibrated. Then, pass the concentrated and buffer-exchanged cell supernatant through the chromatography column at a flow rate of 1 mL / min, allowing the impurity proteins to bind to the chromatography column, and collect the flow-through fraction. After all the supernatant has passed through the chromatography column, rinse the chromatography column with 5 - 10 column volumes of affinity chromatography equilibration buffer until the UV detection line is stable. Finally, rinse the chromatography column successively with elution buffers containing 300 mM, 500 mM, and 1 M NaCl, and subject the sample collected in the flow-through to SDS-PAGE analysis after sample preparation. Further purification is carried out using a SuperoseTM 6 Increase 10 / 300 GL gel filtration chromatography column. The sample obtained from anion exchange chromatography is concentrated to less than 1000 μl using a 100 kDa ultrafiltration concentrator tube and then transferred to a 1.5 mL centrifuge tube. After centrifugation at 12000 rpm and 4 °C for 20 min, and ensuring that no precipitate is aspirated, transfer the sample to a new 1.5 mL centrifuge tube. Centrifuge at 12000 rpm and 4 °C for 20 min to remove air bubbles from the sample and prepare for loading. Equilibrate the SuperoseTM 6 Increase 10 / 300 GL gel filtration chromatography column with 20 mM Tris / 150 mM NaCl at a maximum flow rate of 0.5 mL / min until the UV detection line is stable, and load the sample using a 1 mL loop. After loading, rinse the chromatography column with 20 mM Tris / 150 mM NaCl equilibration buffer at a flow rate of 0.5 mL / min, set peak collection, and collect the samples at the elution peaks. Subject the collected samples to preliminary SDS-PAGE analysis after sample preparation:

[0347] 1) Take 20 μl of the purified sample described above, add 5 μl of 5× protein loading buffer, and heat at 100 °C for 3 min;

[0348] 2) Take 10 μl of the supernatant for SDS-PAGE electrophoresis;

[0349] 3) Stain with Coomassie Brilliant Blue;

[0350] 4) Decolorize with decolorizing solution and observe.

[0351] The identification results are as Figure 27 shown, Figure 27Results: Four virus-like particle vaccines, namely DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, showed a single band between 70 and 55 kDa in SDS-PAGE gel electrophoresis, which was consistent with the expectation, indicating a high purity of the samples.

[0352] Example 21: Characterization of Dengue Virus-Like Particle Vaccine Using Transmission Electron Microscopy

[0353] In this example, the morphology of the dengue virus-like particles prepared in Example 20 was observed and detected by transmission electron microscopy. The specific operation steps are as follows:

[0354] Prepare copper grids, phosphotungstic acid staining solution, filter paper, and sample boxes.

[0355] 1) Preparation of negatively stained samples. First, glow discharge the copper grids. A glow discharge instrument is usually used to hydrophilize the carbon-coated copper grids.

[0356] 2) After glow discharging the copper grids, pick them up with sharp forceps and fix the forceps with long tail clips.

[0357] 3) Drop 6.5 μl of the sample onto the copper grid and let it stand for one minute, then absorb it with filter paper.

[0358] 4) Drop 6.5 μl of phosphotungstic acid onto the copper grid and immediately absorb it with filter paper.

[0359] 5) Drop 6.5 μl of phosphotungstic acid onto the copper grid, let it stand for one minute, and absorb the excess staining solution with a wet filter paper.

[0360] 6) Observe under the electron microscope after drying.

[0361] After negative staining with phosphotungstic acid of the samples purified by chromatography column, the samples were observed and analyzed by a 100 kV electron microscope. The electron microscope results are as Figures 28-31 shown. The negative staining results of DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3 virus-like particles showed that a large number of uniform virus-like particles with a diameter of about 30 nm could be observed in the field of view.

[0362] Example 22: Immunogenicity Detection of Monovalent Dengue Virus Recombinant Protein Vaccine

[0363] Female 6 - 8 - week - old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the vaccine immunization experiment; the experiment was divided into a monovalent dengue virus recombinant protein vaccine immunization group and a negative control group (normal saline, as the Sham group), with 5 mice in each group; for each immunization group, a placebo (or the dengue virus - like particle protein prepared in Example 20 was respectively mixed with a combined adjuvant composed of Al(OH)3 (Invivogen, vac - alu - 250) and CpG 1826 (Invivogen, vac - 1826 - 1)) was intramuscularly injected on day 0 and day 21, the injection dose was 12 μg / dose, and blood was collected 2 weeks after the booster immunization. The serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at - 80°C for later use. The method for detecting the serum neutralizing antibody titer was referred to Example 7.

[0364] The results of the neutralizing antibody titer detection are as Figure 32 shown, which shows that no neutralizing antibody could be detected in the serum of the Sham group, and relatively high neutralizing antibody titers against their own viruses could be detected in all the dengue virus recombinant protein vaccines of the mutants. The neutralizing antibody titers were between 4,000 - 20,000, indicating that the above - mentioned dengue virus recombinant protein vaccines had good immunogenicity.

[0365] Example 23: Immunogenicity Detection of Quadrivalent Dengue Recombinant Protein Vaccine

[0366] Female 6 - 8 - week - old BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the vaccine immunization experiment; the experiment was divided into a quadrivalent dengue virus recombinant protein vaccine group (tVLP) and a negative control group (i.e., the Sham group), with 5 mice in each group. The preparation method of the tVLP vaccine was as follows: The four virus - like particles DV1 - EM3 - 1, DV2 - EM3 - 1, DV3 - EM3, and DV4 - EM3 prepared in Example 20 were mixed in equal proportion according to the mass ratio of 1:1:1:1, 2.5 μg / dose for each, a total of 10 μg / dose, and mixed with a combined adjuvant composed of Al(OH)3 (Invivogen, vac - alu - 250) and CpG 1826 (Invivogen, vac - 1826 - 1). Placebo or vaccine was intramuscularly injected on day 0 and day 21 respectively. Blood was collected 2 weeks after the booster immunization, the serum was separated by centrifugation, the serum was inactivated by heating at 56°C for 30 minutes, and then stored at - 80°C for later use. The method for detecting serum neutralizing antibody was referred to the Example.

[0367] The results of the neutralization experiment are as Figure 33As shown, it shows that no neutralizing antibodies against dengue virus of four serotypes can be detected in the serum of the Sham group, and the tVLP vaccine can induce a balanced and strong neutralizing antibody immune response against dengue virus of four serotypes, with the neutralizing antibody titer being 1,100 - 4,000, indicating that the tetravalent dengue virus recombinant protein vaccine composed of mutant virus-like particles has good immunogenicity.

[0368] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant antigen, wherein the recombinant antigen has the full-length sequence of the E protein of Zika virus or Dengue virus, and the E protein has site mutations selected from the following: A site mutation at position G106 in the FL fusion loop region; Double-site mutations at positions W101 and G106 in the FL fusion loop region; Double-site mutations at positions W101 and G106 in the FL fusion loop region and a mutation at the 125th amino acid in the non-FL fusion loop region; Double-site mutations at positions G102 and G106 in the FL fusion loop region; Double-site mutations at positions N103 and G106 in the FL fusion loop region; Triple-site mutations at positions W101, N103 and G106 in the FL fusion loop region.

2. The recombinant antigen according to claim 1, wherein The site mutation at position G106 in the FL fusion loop region is: G106V mutation; And / or, the double-site mutations at positions W101 and G106 in the FL fusion loop region are selected from: (1) W101R and G106V, (2) W101G and G106V, and (3) W101L and G106V; And / or, the double-site mutations at positions W101 and G106 in the FL fusion loop region and the mutation at the 125th amino acid in the non-FL fusion loop region are: double-site mutations at positions W101R and G106V in the FL fusion loop region and a mutation of the 125th amino acid in the non-fusion loop region to valine; And / or, the double-site mutations at positions G102 and G106 in the FL fusion loop region are: double-site mutations at positions G102R and G106V; And / or, the double-site mutations at positions N103 and G106 in the FL fusion loop region are selected from: (1) N103T and G106V, (2) N103H and G106V, (3) N103K and G106V, (4) N103P and G106V, and (5) N103Y and G106V; And / or, the triple-site mutations at positions W101, N103 and G106 in the FL fusion loop region are selected from: (1) W101G, N103T and G106V, and (2) W101G, N103K and G106V.

3. The recombinant antigen according to claim 2, wherein, The recombinant antigen has the full-length sequence of the E protein of Dengue virus type 1, and the E protein has site mutations selected from the following: G106V mutation; Double-site mutations at positions W101R and G106V; Double-site mutations at positions W101G and G106V; Double-site mutations at positions W101L and G106V; Triple-site mutations at positions W101R, G106V and L125V; Double-site mutations at positions G102R and G106V; Double-site mutations at positions N103T and G106V; Double-site mutations at positions N103H and G106V; Double-site mutations at positions N103K and G106V; Double-site mutations at positions N103P and G106V; Double-site mutations at positions N103Y and G106V; Triple-site mutations at positions W101G, N103T and G106V; Triple-site mutations at positions W101G, N103K and G106V.

4. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of Dengue virus type 2, and the E protein has site mutations selected from the following: G106V mutation; Double-site mutations at positions W101R and G106V; Double-site mutations at positions W101G and G106V; Double-site mutations at positions W101L and G106V; Three-site mutations of W101R, G106V, and M125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

5. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, and the E protein has site mutations selected from the following: G106V mutation; Two-site mutations of W101R and G106V; Two-site mutations of W101G and G106V; Two-site mutations of W101L and G106V; Three-site mutations of W101R, G106V, and I125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

6. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, and the E protein has site mutations selected from the following: G106V mutation; Two-site mutations of W101R and G106V; Two-site mutations of W101G and G106V; Two-site mutations of W101L and G106V; Three-site mutations of W101R, G106V, and I125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

7. The recombinant antigen according to any one of claims 1-6, characterized in that, When the recombinant antigen has the full-length sequence of the E protein of Zika virus, the recombinant antigen further includes the full-length or partial prM protein sequence or M protein sequence of Zika virus.

8. The recombinant antigen according to any one of claims 1-6, characterized in that, When the recombinant antigen has the full-length sequence of the E protein of dengue virus, the recombinant antigen further includes the full-length or partial prM protein sequence or M protein sequence of the corresponding serotype of dengue virus; Preferably, when the recombinant antigen has the full-length E protein sequence of dengue virus type 1, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 2-14; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 2, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 16-28; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 3, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 30-42; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 4, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 44-56.

9. The preparation method of the recombinant antigen according to any one of claims 1-8, characterized in that, The preparation method comprises the following steps: Adding a Kozak sequence and the coding sequence of a signal peptide to the 5' end of the nucleotide sequence encoding the recombinant antigen according to any one of claims 1-8, adding the coding sequence of a histidine tag and a stop codon to the 3' end, performing clone expression, screening correct recombinants, then transfecting expression system cells for expression, collecting the cell culture supernatant, and isolating the recombinant antigen therefrom.

10. The preparation method according to claim 9, characterized in that, The expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells; Optionally, the mammalian cells are HEK293T cells, 293F series cells or CHO cells; further optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells; Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells; Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom; Optionally, the bacterial cells are Escherichia coli cells.

11. A polynucleotide encoding the recombinant antigen according to any one of claims 1-8.

12. The polynucleotide according to claim 11, wherein The polynucleotide is DNA or mRNA, preferably mRNA; Preferably, the polynucleotide is DNA having a sequence shown in any one of SEQ ID NO: 62-113, or the mRNA corresponding to the DNA.

13. An expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus comprising the polynucleotide according to claim 11 or 12.

14. A vaccine composition comprising the recombinant antigen according to any one of claims 1-8, or the polynucleotide according to claim 11 or 12, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus according to claim 13 as an active ingredient.

15. The vaccine composition according to claim 14, characterized in that, It is a recombinant protein vaccine, which comprises the recombinant antigen according to any one of claims 1-8 and an adjuvant; Preferably, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, CpG adjuvant, QS-21-containing adjuvant, AS series adjuvants and nanoparticle adjuvant.

16. The vaccine composition according to claim 15, characterized in that, It is a DNA vaccine, and the DNA vaccine comprises: (i) A eukaryotic expression vector; and (ii) A DNA sequence encoding the recombinant antigen as described in any one of claims 1-8, which is constructed into the eukaryotic expression vector; Preferably, the DNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is a DNA sequence shown in any one of SEQ ID NO: 62-113; And / or, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

17. The vaccine composition according to claim 14, characterized in that, It is an mRNA vaccine, and the mRNA vaccine comprises: (I) An mRNA sequence encoding the recombinant antigen as described in any one of claims 1-8; and (II) Lipid nanoparticles; Preferably, the mRNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is an mRNA sequence corresponding to the DNA sequence shown in any one of SEQ ID NO: 62-113.

18. The vaccine composition according to claim 14, characterized in that, It is a viral vector vaccine, which comprises: (1) A viral backbone vector; and (2) A DNA sequence encoding the recombinant antigen as described in any one of claims 1-8, which is constructed into the viral backbone vector; Preferably, the DNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is a DNA sequence shown in any one of SEQ ID NO: 62-113; And / or, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, lentivirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

19. The vaccine composition according to any one of claims 14-18, characterized in that, The vaccine composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation; Preferably, the nasal spray is selected from aerosols, sprays and powder aerosols; Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; More preferably, the tablet is a sublingual tablet; More preferably, the granule is a fine granule; More preferably, the powder is a powder; More preferably, the pill is a small pill; Preferably, the parenteral preparation is a transdermal agent, ointment, plaster, external liquid agent, injectable preparation; more preferably, the injectable preparation is a pushable preparation.

20. Use of the recombinant antigen as described in any one of claims 1-8, the polynucleotide as described in claim 11 or 12, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus as described in claim 13 in the preparation of a drug for detecting, preventing and / or treating Zika virus or dengue virus infection; Preferably, the drug is a vaccine.

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